WO2015009558A1 - Compositions et procédés de biosynthèse de vanilline ou d'un bêta-d-glucoside de vanilline - Google Patents

Compositions et procédés de biosynthèse de vanilline ou d'un bêta-d-glucoside de vanilline Download PDF

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WO2015009558A1
WO2015009558A1 PCT/US2014/046315 US2014046315W WO2015009558A1 WO 2015009558 A1 WO2015009558 A1 WO 2015009558A1 US 2014046315 W US2014046315 W US 2014046315W WO 2015009558 A1 WO2015009558 A1 WO 2015009558A1
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vanillin
polypeptide
nucleic acid
recombinant host
acid encoding
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PCT/US2014/046315
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English (en)
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Esben Halkjaer HANSEN
Swee Chuang Lim HALLWYL
Klavs Riishede HANSEN
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International Flavors & Fragrances Inc.
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Application filed by International Flavors & Fragrances Inc. filed Critical International Flavors & Fragrances Inc.
Priority to ES14826331T priority Critical patent/ES2950245T3/es
Priority to US14/905,228 priority patent/US10000782B2/en
Priority to EP14826331.2A priority patent/EP3022291B1/fr
Publication of WO2015009558A1 publication Critical patent/WO2015009558A1/fr
Priority to US15/980,190 priority patent/US10066252B1/en

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/46—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C47/00—Compounds having —CHO groups
    • C07C47/52—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings
    • C07C47/575—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings containing ether groups, groups, groups, or groups
    • C07C47/58—Vanillin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
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    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01001—Alcohol dehydrogenase (1.1.1.1)

Definitions

  • Vanillin is one of the world's most important flavor compounds with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (Vanilla planifolia) , but most of the world's vanillin is synthesized from petrochemicals or wood pulp lignins. Production of natural vanillin from the vanilla pod is a laborious and slow process, which requires hand pollination of the flowers and a 1-6 month curing process of the harvested green vanilla pods (Ramachandra & Ravishankar (2000) J. Sci . Food Agric . 80:289-304) .
  • This invention provides a recombinant host cell having the following characteristics: the recombinant host cell produces vanillin and/or vanillin beta-D-glucoside ; and the recombinant host cell has reduced production or activity of a first alcohol dehydrogenase and reduced production of one or more second alcohol dehydrogenases, one or more aldehyde reductases, or a combination thereof.
  • the first alcohol dehydrogenase is Alcohol Dehydrogenase 6 (ADH6) .
  • the one or more second alcohol dehydrogenases include Alcohol Dehydrogenase 7 (ADH7) , Genes de Respuesta a Estres 2 (GRE2) , or an ortholog thereof.
  • the aldehyde reductase includes Aldehyde Reductase Intermediate 1 (ARI1) , Aldehyde Reductase YGL039W, or an ortholog thereof.
  • the recombinant host cell further includes a nucleic acid encoding an AROM polypeptide, a nucleic acid encoding a catechol -O- methyltransferase (COMT) polypeptide, a nucleic acid encoding a 3 -dehydroshikimate dehydratase (3DSD) polypeptide, a nucleic acid encoding an aromatic carboxylic acid reductase (ACAR) polypeptide, a nucleic acid encoding a phosphopantetheine transferase (PPTase) polypeptide, a nucleic acid encoding an uridine 5 ' -diphosphoglucosyl transferase (UGT) polypeptide and/or a nucleic acid encoding an A
  • a consumable e.g., a food product, pharmaceutical composition, a dietary supplement, a nutraceutical , a dental hygienic composition, a tabletop sweetener, or a cosmetic product containing the extract.
  • Figure 1 is a schematic of de novo biosynthesis of vanillin (4) and outline of the different vanillin catabolites and metabolic side products, i.e., dehydroshikimic acid (1) , protocatechuic acid (2) , protocatechuic aldehyde (3) , vanillic acid (5) , protocatechuic alcohol (6) , vanillyl alcohol (7) , and vanillin ⁇ -D-glucoside (8) , found in an organism expressing 3DSD, ACAR, OMT, and UGT and a phophopantheteine transferase (PPTase) .
  • Open arrows show primary metabolic reactions in yeast; black arrows show enzyme reactions introduced by metabolic engineering; diagonally striped arrows show undesired inherent yeast metabolic reactions.
  • Figure 2 shows the production of vanillyl alcohol glucoside, vanillyl alcohol, vanillin ⁇ -D-glucoside and vanillin in a strain lacking one or more functional alcohol dehydrogenases .
  • Figure 3 shows the production of vanillyl alcohol glucoside, vanillyl alcohol, vanillin ⁇ -D-glucoside and vanillin in a strain lacking gre2 (EFSC2055 gre2 ) or gre2 and aril (EFSC2055 gre2 aril) as compared to the parental strain EFSC2055. *, p ⁇ 0.01.
  • This invention is based on the discovery that knocking out certain alcohol dehydrogenases and/or aldehyde reductases, or similar enzymes, lowers the amount of vanillic alcohol that is formed as a byproduct. See Figures 1 and 2. This is of commercial importance because the presence of alcohol creates inefficiencies in certain steps of the downstream purification of vanillin or vanillin glucoside if the alcohol is allowed to accumulate.
  • the present invention is a recombinant host that is capable of producing vanillin or vanillin glucoside, but fails to produce, or has reduced production of, one or more alcohol dehydrogenases and/or one or more aldehyde reductases.
  • a recombinant host that produces or is capable of producing vanillin or vanillin glucoside is a host cell that expresses the necessary biosynthetic enzymes to produce vanillin or vanillin glucoside from a primary substrate, e.g., glucose, or from an intermediate molecule, e.g., dehydroshikimic acid, protocatechuic acid, protocatechuic aldehyde, or vanillic acid. See Figure 1.
  • a recombinant host that fails to produce an enzyme, has reduced production of an enzyme, or lacks a functional enzyme includes an organism that have been recombinantly modified such that the gene encoding the enzyme is knocked out, an organism with one or more point mutations in the enzyme which reduces or diminishes enzyme activity, or an organism wherein the promoter of the gene encoding the enzyme has been modified or removed so that the enzyme is not expressed or expressed at a reduced level compared to a wild-type organism.
  • Modifications that may be used to reduce or eliminate expression of a target enzyme are disruptions that include, but are not limited to, deletion of the entire gene or a portion of the gene encoding an enzyme; inserting a DNA fragment into a gene encoding the enzyme (in either the promoter or coding region) so that the enzyme is not expressed or expressed at lower levels; introducing a mutation into the coding region for the enzyme, which adds a stop codon or frame shift such that a functional enzyme is not expressed; and introducing one or more mutations into the coding region of an enzyme to alter amino acids so that a non-functional or a less enzymatically active enzyme is expressed.
  • expression of an enzyme may be blocked by expression of an antisense RNA or an interfering RNA, and constructs may be introduced that result in cosuppression .
  • the synthesis or stability of the transcript may be lessened by mutation.
  • the efficiency by which an enzyme is translated from mRNA may be modulated by mutation. All of these methods may be readily practiced by one skilled in the art making use of the known sequences encoding the alcohol dehydrogenases and/or aldehyde reductases of this invention.
  • Alcohol dehydrogenase and aldehyde reductase sequences from a variety of organisms are well-known in the art and the selection of the target gene(s) will be dependent upon the host selected.
  • Representative alcohol dehydrogenase (ADH) and aldehyde reductase sequences, which may be targeted in accordance with the present invention are listed in Table 1.
  • One skilled in the art may choose specific modification strategies to eliminate or lower the expression of an alcohol dehydrogenase and/or aldehyde reductase as desired to facilitate vanillin and/or vanillin glucoside production. TABLE 1
  • the recombinant host cells has reduced production or activity of a first alcohol dehydrogenase and reduced production of one or more second alcohol dehydrogenases, one or more aldehyde reductases, or a combination thereof.
  • the first alcohol dehydrogenase is ADH6 or an ortholog thereof, e.g., CAD9, CAD3 or CAD2 from A. thaliana.
  • the one or more second alcohol dehydrogenases are selected from the group of ADH7, GRE2 (Genes de Respuesta a Estres 2), or an ortholog thereof, e.g., AT1G51410 or AT5G19440; and the aldehyde reductase is selected from the group of ARI1 (Aldehyde Reductase Intermediate 1) , Aldehyde Reductase YGL039 , or an ortholog thereof, e.g., SPAC513.07 or YDR541C) .
  • DNA sequences surrounding one or more of the above- referenced sequence are also useful in some modification procedures and are available for yeasts such as for Saccharomycse cerevisiae in the complete genome sequence coordinated by NCBI (National Center for Biotechnology Information) with identifying BioProject Nos . PRJNA128, PRJNA13838, PRJNA43747, PRJNA48559, PRJNA52955, PRJNA48569, PRJNA39317. Additional examples of yeast genomic sequences include that of Schizosaccharomyces pombe, which is included in BioProject Nos . PRJNA127, PRJNA13836, and PRJNA20755.
  • Genomic sequences of plants are also known in the art and the genomic sequence of Arabidopsis thaliana is included in BioProject Nos. PRJNA116, PRJNA10719, PRJNA13190, and PRJNA30811. Other genomic sequences can be readily found by one of skill in the art in publicly available databases.
  • DNA sequences surrounding an alcohol dehydrogenase or aldehyde reductase coding sequence are useful for modification methods using homologous recombination.
  • sequences flanking the gene of interest are placed on either side of a selectable marker gene to mediate homologous recombination whereby the marker gene replaces the gene of interest.
  • partial gene sequences and flanking sequences bounding a selectable marker gene may be used to mediate homologous recombination whereby the marker gene replaces a portion of the target gene.
  • the selectable marker may be bounded by site-specific recombination sites, so that following expression of the corresponding site-specific recombinase, the resistance gene is excised from the gene of interest without reactivating the latter.
  • the site-specific recombination leaves behind a recombination site which disrupts expression of the alcohol dehydrogenase or aldehyde reductase.
  • the homologous recombination vector may be constructed to also leave a deletion in the gene of interest following excision of the selectable marker, as is well known to one skilled in the art.
  • Deletions may be made using mitotic recombination as described in Wach, et al . ((1994) Yeast 10:1793-1808) .
  • This method involves preparing a DNA fragment that contains a selectable marker between genomic regions that may be as short as 20 bp, and which bound a target DNA sequence.
  • This DNA fragment can be prepared by PCR amplification of the selectable marker gene using as primers oligonucleotides that hybridize to the ends of the marker gene and that include the genomic regions that can recombine with the yeast genome.
  • the linear DNA fragment can be efficiently transformed into yeast and recombined into the genome resulting in gene replacement including with deletion of the target DNA sequence.
  • promoter replacement methods may be used to exchange the endogenous transcriptional control elements allowing another means to modulate expression such as described in Mnaimneh, et al . ((2004) Cell 118 (1) : 31-44) .
  • Hosts cells of use in this invention include any organism capable of producing vanillin and/or vanillin glucoside either naturally or synthetically, e.g., by recombinant expression of one or more genes of the vanillin and/or vanillin glucoside biosynthetic pathway ( Figure 1) .
  • a number of prokaryotes and eukaryotes are suitable for use in constructing the recombinant microorganisms described herein, e.g., gram-negative bacteria, gram-positive bacteria, yeast or other fungi.
  • a species and strain selected for use as a vanillin and/or vanillin glucoside production strain is first analyzed to determine which production genes are endogenous to the strain and which genes are not present. Genes for which an endogenous counterpart is not present in the strain are assembled in one or more recombinant constructs, which are then transformed into the strain in order to supply the missing function (s) .
  • Exemplary prokaryotic and eukaryotic species are described in more detail below. However, it will be appreciated that other species may be suitable.
  • suitable species may be in a genus Agaricus, Aspergillus, Bacillus, Candida, Corynehacterium,
  • Schizosaccharomyces, Sphaceloma, Xanthophyllomyces Yarrowia and Lactobacillus Exemplary species from such genera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporiu , Pichia pastoris,
  • a microorganism can be an Ascomycete such as Gibberella fujikuroi , Kluyveromyces lactis,
  • a microorganism can be a prokaryote such as Escherichia coli, Rhodobacter sphaeroides, or Rhodobacter capsulatus . It will be appreciated that certain microorganisms can be used to screen and test genes of interest in a high throughput manner, while other microorganisms with desired productivity or growth characteristics can be used for large-scale production of vanillin beta-D-glucoside.
  • S. cerevisiae which may be recombinantly engineered as described herein.
  • S. cerevisiae is a widely used chassis organism in synthetic biology, and can be used as the recombinant microorganism platform.
  • the VG4 strain of S. cerevisiae (Brochado, et al . (2010) Microb. Cell Fact. 9:84) is particularly useful.
  • VG4 has the genotype of pdclAgdh1Z1 ⁇ GDH2.
  • Aspergillus species such as A. oryzae, A. niger and A. sojae are widely used microorganisms in food production, and can also be used as the recombinant microorganism platform.
  • the recombinant host may be Aspergillus spp. Nucleotide sequences are available for genomes of A. nidulans, A. fumigatus, A. oryzae, A. clavatus, A. flavus, A. niger, and A. terreus, allowing rational design and modification of endogenous pathways to enhance flux and increase product yield. Metabolic models have been developed for Aspergillus, as well as transcriptomic studies and proteomics studies.
  • A. niger is cultured for the industrial production of a number of food ingredients such as citric acid and gluconic acid, and thus species such as A. niger are generally suitable for the production of food ingredients such as vanillin and vanillin glucoside .
  • E. coli another widely used platform organism in synthetic biology, can also be used as the recombinant microorganism platform.
  • the recombinant host may be E. coli.
  • Saccharo yces Similar to Saccharo yces , there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for Saccharomyces can be used to make recombinant E. coli microorganisms .
  • Rhodobacter can be used as the recombinant microorganism platform.
  • the recombinant host may be Rhodobacter spp. Similar to E. coli, there are libraries of mutants available as well as suitable plasmid vectors, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for E. coli can be used to make recombinant Rhodobacter microorganisms .
  • Physcomitrella mosses when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera is becoming an important type of cell for production of plant secondary metabolites, which can be difficult to produce in other types of cells.
  • the recombinant host may be a Physcomitrella spp.
  • the recombinant host is a plant or plant cells that includes the one or more genes of the vanillin and/or vanillin glucoside biosynthetic pathway.
  • a plant or plant cell can be modified to express the vanillin and/or vanillin glucoside biosynthetic pathway with a concurrent knockout of one or more alcohol dehydrogenases and/or aldehyde reductases.
  • the plant or plant cells can be stably transformed to retain the introduced nucleic acid with each cell division.
  • a plant or plant cell can also be transiently transformed such that the heterologous nucleic acid is not integrated into its genome.
  • Transiently transformed cells typically lose all or some portion of the introduced nucleic acid with each cell division such that the introduced nucleic acid cannot be detected in daughter cells after a sufficient number of cell divisions. Both transiently transformed and stably transformed transgenic plants and plant cells can be useful in the methods described herein.
  • Transgenic plant cells used in methods described herein can constitute part or all of a whole plant. Such plants can be grown in a manner suitable for the species under consideration, either in a growth chamber, a greenhouse, or in a field. Transgenic plants can be bred as desired for a particular purpose, e.g., to introduce a heterologous nucleic acid, for example a recombinant nucleic acid construct into other lines, to transfer a heterologous nucleic acid to other species, or for further selection of other desirable traits. Alternatively, transgenic plants can be propagated vegetatively for those species amenable to such techniques. As used herein, a transgenic plant also refers to progeny of an initial transgenic plant provided the progeny inherits the transgene. Seeds produced by a transgenic plant can be grown and then selfed (or outcrossed and selfed) to obtain seeds homozygous for the nucleic acid construct .
  • Transgenic plants can be grown in suspension culture, or tissue or organ culture.
  • solid and/or liquid tissue culture techniques can be used.
  • transgenic plant cells can be placed directly onto the medium or can be placed onto a filter that is then placed in contact with the medium.
  • transgenic plant cells can be placed onto a flotation device, e.g., a porous membrane that contacts the liquid medium.
  • a reporter sequence encoding a reporter polypeptide having a reporter activity can be included in the transformation procedure and an assay for reporter activity or expression can be performed at a suitable time after transformation.
  • a suitable time for conducting the assay typically is about 1-21 days after transformation, e.g., about 1-14 days, about 1-7 days, or about 1-3 days.
  • the use of transient assays is particularly convenient for rapid analysis in different species, or to confirm expression of a heterologous polypeptide whose expression has not previously been confirmed in particular recipient cells.
  • nucleic acids into monocotyledonous and dicotyledonous plants are known in the art, and include, without limitation, Agrobacterium- mediated transformation, viral vector-mediated transformation, electroporation and particle gun transformation; see U.S. Patent Nos . 5,538,880; 5,204,253; 6,329,571; and 6,013,863. If a cell or cultured tissue is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.
  • a population of transgenic plants can be screened and/or selected for those members of the population that have a trait or phenotype conferred by expression of the transgene .
  • a population of progeny of a single transformation event can be screened for those plants having a desired level of expression of a polypeptide or nucleic acid described herein. Physical and biochemical methods can be used to identify expression levels.
  • a population of plants with independent transformation events can be screened for those plants having a desired trait, such as production of vanillin glucoside and lack of vanillic alcohol production. Selection and/or screening can be carried out over one or more generations, and/or in more than one geographic location.
  • transgenic plants can be grown and selected under conditions which induce a desired phenotype or are otherwise necessary to produce a desired phenotype in a transgenic plant.
  • selection and/or screening can be applied during a particular developmental stage in which the phenotype is expected to be exhibited by the plant .
  • the recombinant host cell can naturally or recombinantly express genes encoding an AROM (Arom Multifunctional Enzyme) , OMT (O-methyltransferase) , COMT
  • Recombinant expression means that the genome of a host cell has been augmented through the introduction of one or more recombinant genes, which include regulatory sequences that facilitate the transcription and translation of a protein of interest. While embodiments include stable introduction of recombinant genes into the host genome, autonomous or replicative plasmids or vectors can also be used within the scope of this invention. Moreover, the present invention can be practiced using a low copy number, e.g., a single copy, or high copy number (as exemplified herein) plasmid or vector.
  • the introduced recombinant gene is not originally resident in the host that is the recipient of the recombinant gene, but it is within the scope of the invention to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene.
  • the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis.
  • Suitable recombinant hosts include microorganisms, plant cells, and plants.
  • recombinant gene refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host. "Introduced,” or “augmented” in this context, is known in the art to mean introduced or augmented by the hand of man. Thus, a recombinant gene may be a DNA sequence from another species, or may be a DNA sequence that originated from or is present in the same species, but has been incorporated into a host by recombinant methods to form a recombinant host .
  • a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA.
  • a recombinant gene encoding a polypeptide described herein includes the coding sequence for that polypeptide, operably linked, in sense orientation, to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired.
  • a coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence.
  • the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene .
  • the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid.
  • heterologous nucleic acid refers to a nucleic acid introduced into a recombinant host, wherein said nucleic acid is not naturally present in said host.
  • the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals.
  • the coding sequence is a sequence that is native to the host and is being reintroduced into that organism.
  • a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non- native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct.
  • stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found.
  • regulatory region refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3" untranslated regions (UTRs) , transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof.
  • a regulatory region typically includes at least a core (basal) promoter.
  • a regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR) .
  • a regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence.
  • the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter.
  • a regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site .
  • regulatory regions The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region may be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
  • One or more genes can be combined in a recombinant nucleic acid construct in "modules" useful for a discrete aspect of vanillin and/or vanillin glucoside production.
  • modules useful for a discrete aspect of vanillin and/or vanillin glucoside production.
  • Combining a plurality of genes or heterologous nucleic acids in a module facilitates the use of the module in a variety of species.
  • a vanillin gene cluster can be combined such that each coding sequence is operably linked to a separate regulatory region, to form a vanillin module for production in eukaryotic organisms.
  • the module can express a polycistronic message for production of vanillin and/or vanillin glucoside in prokaryotic hosts such as species of Rodobacter, E. coll, Bacillus or Lactobacillus.
  • a recombinant construct typically also contains an origin of replication, and one or more selectable markers for maintenance of the construct in appropriate species .
  • codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for that host (e.g., microorganism) .
  • these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs .
  • recombinant hosts can express one or more enzymes involved in the biosynthesis of the vanillin or vanillin glucoside, as well as additional genes or biosynthetic modules that improve efficiency with which energy and carbon sources are converted to vanillin and its glucoside, and/or to enhance productivity from the cell culture or plant.
  • the recombinant host endogenously ore recombinantly expresses genes encoding A OM, COMT, 3DSD, ACAR, UGT, and/or PPTase .
  • AROM is a penta- functional enzyme complex encoded in yeast by the AROl gene.
  • the gene is 4764 bp long and encodes a corresponding polypeptide 1588 amino acids in length.
  • AROM performs five consecutive enzymatic conversions, i.e., converting DAHP (3 -deoxy-D-arabino- heptulosonic acid-7-phosphate) into 3-DHQ (3- dehydroquinate) , which is converted to 3-DHS (3- dehydroshikimic acid) , which is converted to shikimate, which is converted to shikimate-3-P (shikimate 3- phosphate) , which is converted into EPSP (5- enolpyruvylskimate 3 -phosphate) , all en route to cellular biosynthesis of the aromatic amino acids tyrosine, tryptophan and phenylalanine.
  • the AROM enzyme possesses at least four of the five enzymatic activities of the S. cerevisiae AROM polypeptide, i.e., 3 -dehydroquinate dehydratase activity, 3 -dehydroquinate synthase activity, 3 -phosphoshikimate 1- carboxyvinyltransferase activity, shikimate 3 -dehydrogenase
  • Non-limiting examples of AROM polypeptides include the Saccharomyces cerevisiae polypeptide available under GENBANK Accession No. X06077; the Schizosaccharo yces pombe polypeptide available under GENBANK Accession No. NP_594681.1; Schizosaccharomyces japonicas polypeptide available under GENBANK Accession No. XP_00217162 ; Neurospora crassa polypeptide available under GENBANK Accession No. XP_956000; and the Yarrowia lipolytica polypeptide available under GENBANK Accession No. XP_505337.
  • the AROM polypeptide is a mutant AROM polypeptide with decreased shikimate dehydrogenase activity. When expressed in a recombinant host, the mutant AROM polypeptide redirects metabolic flux from aromatic amino acid production to vanillin precursor production, i.e., 3-DHS. See WO 2013/022881.
  • the mutant AROM polypeptide described herein can have one or more modifications in domain 5 ⁇ e.g., a substitution of one or more amino acids, a deletion of one or more amino acids, insertions of one or more amino acids, or combinations of substitutions, deletions, and insertions) .
  • a modified AROM polypeptide is fused to a polypeptide catalyzing the first committed step of vanillin biosynthesis, 3DSD.
  • a polypeptide having 3DSD activity and that is suitable for use in a fusion polypeptide includes the 3DSD polypeptide from Podospora pauciseta, Ustilago maydis, Rhodoicoccus jostii, Acinetobacter sp., Aspergillus niger or Neurospora crassa. See, GENBANK Accession Nos . CAD60599) , XP_001905369.1 , XP_761560.1, ABG93191.1, AAC37159.1, and XM_001392464.
  • COMT polypeptides of use in this invention include COMT polypeptides in the family classified under EC number 2.1.1.6, such as the Homo sapiens (Hs) polypeptide available under GENBANK Accession No. NM_000754; an Arabidopsis thaliana polypeptide available under GENBANK Accession No. AY062837 or a Fragaria x ananassa (strawberry) polypeptide available under GENBANK Accession No. AF220491.
  • Human COMT polypeptide exists as several variants and the COMT polypeptide may be any of these variants.
  • COMT polypeptides of use in this invention include, but are not limited to, those isolated from Pan troglodytes (GENBANK Accession No. XP_514984) , Macaca ulatta (GENBANK Accession No. AFJ70145) , Equus caballus (GENBANK Accession No. NP_001075303) , Canis lupus familiaris (GENBANK Accession No. AAR20324) , Cricetulus griseus (GENBANK Accession No. EGV97595) , Sus scrota (GENBANK Accession No. NP_001182259) , and Bos taurus (GENBANK Accession No. NP_001095787) .
  • COMT polypeptides from plant and microorganism sources include, but are not limited to, those isolated from Rosa chinensis (GENBANK Accession No. CAD29457) , Prunus dulcis (GENBANK Accession No. CAA58218) , Gossypium hirsutum (GENBANK Accession No. ACT32028) , Jatropha curcas (GENBANK Accession No. ACT87981) , Eucalyptus ca aldulensis (ADB82906) , Candida orthopsilosis (GENBANK Accession No. CCG25047) , Pichia stipitis (GENBANK Accession No.
  • the COMT polypeptide of the invention is obtained from Phytophthera infestans (GENBANK Accession No. XP_00289921 ) , Catharanthus roseus (GENBANK Accession No. EGS21863), Yarrowia lipolytica (GENBANK Accession No. XP_500451) , Ciona intestinalis (GENBANK Accession No. XP__002121420 or XP_002131313 ) , Capsasproa owczarzaki (GENBANK Accession No.
  • NP_031770 Crenarchaeote (GENBANK Accession No. ABZ07345) , Mycobacterium vanbaleeni (GENBANK Accession No. ABM14078) , or Schizosaccharomyces pombe (GENBANK Accession No. NP_001018770.
  • mutant COMT polypeptide is used to improve biosynthesis of vanillin beta-D-glucoside .
  • mutant COMT polypeptides can have one or more of the following properties: increased turnover; preferential methylation at the meta (3') position, rather than at the para (4') position such that production of vanillin is favored over isovanillin; or better specificity for the vanillin pathway substrates, protocatechuic acid and protocatechuic aldehyde. See WO 2013/022881.
  • a mutant COMT polypeptide can have one or more mutations (e.g., a substitution of one or more amino acids, a deletion of one or more amino acids, insertions of one or more amino acids, or combinations of substitutions, deletions, and insertions) in, for example, the substrate binding site.
  • a mutant COMT polypeptide can have one or more amino acid substitutions in the substrate binding site of human COMT.
  • a mutant COMT polypeptide is provided, which is capable of catalyzing methylation of an -OH group of protocatechuic acid, wherein said methylation results in generation of at least 4 times more vanillic acid compared to iso-vanillic acid.
  • the mutant COMT polypeptide is capable of catalyzing methylation of an -OH group of protocatechuic aldehyde, wherein said methylation results in generation of at least 4 times more vanillin compared to iso-vanillin .
  • the host harbors a nucleic acid encoding mutant AROM polypeptide and optionally a wild-type COMT polypeptide. In another embodiment, the host of this invention harbors a nucleic acid encoding mutant COMT polypeptide and optionally a wild-type AROM polypeptide. In yet another embodiment, the host of this invention harbors a nucleic acid encoding mutant AROM polypeptide and optionally a mutant COMT polypeptide.
  • a 3DSD polypeptide according to the present invention may be any enzyme with 3 -dehydroshikimate dehydratase activity.
  • the 3DSD polypeptide is an enzyme capable of catalyzing conversion of 3 -dehydro-shikimate to protocatechuate and H 2 0.
  • a 3DSD polypeptide according to the present invention is preferably an enzyme classified under EC 4.2.1.118.
  • a suitable polypeptide having 3DSD activity includes the 3DSD polypeptide made by Podospora pauciseta, Ustilago maydis, Rhodoicoccus jostii, Acinetobacter sp.
  • the recombinant host may include a heterologous nucleic acid encoding the 3DSD polypeptide of Podospora anserina, Ustilago maydis, Rhodoicoccus jostii,
  • ACAR polypeptides are also known in the art.
  • An ACAR polypeptide according to the present invention may be any enzyme having aromatic carboxylic acid reductase activity.
  • the ACAR polypeptide is an enzyme capable of catalyzing conversion protocatechuic acid to protocatechuic aldehyde and/or conversion of vanillic acid to vanillin.
  • An ACAR polypeptide according to the present invention is preferably an enzyme classified under EC 1.2.1.30.
  • a suitable ACAR polypeptide is made by Nocardia sp . See, e.g., GENBANK Accession No. AY495697.
  • the recombinant host may include a heterologous nucleic acid encoding the ACAR polypeptide of Nocardia sp . or a functional homologue thereof sharing at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 98% sequence identity therewith .
  • Suitable PPTase polypeptides are known.
  • a PPTase polypeptide according to the present invention may be any enzyme capable of catalyzing phosphopantetheinylation.
  • the PPTase polypeptide is an enzyme capable of catalyzing phosphopantetheinylation of ACAR.
  • a suitable PPTase polypeptide is made by E. coli, Corynebacterium glutamicu , or Nocardia farcinica . See GENBANK Accession Nos . NP_601186, BAA35224, and YP 120266.
  • the recombinant host may include a heterologous nucleic acid encoding the PPTase polypeptide of E. coli, C. glutamicum, or N. farcinica or a functional homologue of any of the aforementioned sharing at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 98% sequence identity therewith.
  • Vanillin- ⁇ -D-glucoside is the storage form of vanillin found in the vanilla pod. It is non- toxic to most organisms, including yeast, and has a higher solubility in water, as compared to vanillin. In addition, the formation of vanillin- ⁇ -D-glucoside most likely directs the biosynthesis toward vanillin production.
  • UGT72E2 Haansen, et al. (2009) Appl . Environ. Microbiol. 75.-2765-27 40
  • the recombinant host of this invention can also express a UGT polypeptide.
  • a UGT polypeptide may be any UDP-Glucose :Aglycon-Glucosyltransferase .
  • the UGT polypeptides can catalyze the glucosylation of vanillin (i.e., to produce vanillin beta-D-glucoside) .
  • the UGT polypeptide may be a Family 1 glycosyltransferease .
  • Preferred UGT polypeptides according to the invention are classified under EC 2.4.1.
  • Suitable UGT polypeptides include the UGT71C2, UGT72B1, UGT72E2, UGT84A2 , UGT89B1, UGT85B1, and arbutin synthase polypeptides. See, e.g., GENBA K Accession Nos . AC0005496, NM_116337, and NM_126067.
  • the A. thaliana UGT72E2 is particularly useful (see, e.g., Hansen, et al . (2009) supra) .
  • the recombinant host may include a heterologous nucleic acid encoding the UGT71C2, UGT72B1, UGT72E2, UGT84A2, UGT89B1, UGT85B1 , or arbutin synthase or a functional homologue of any of the aforementioned sharing at least 80%, such as at least 85%, for example at least 90%, such as at least 95%, for example at least 98% sequence identity therewith.
  • Other useful UGTs are described in WO 01/40491.
  • VAO enzyme (EC 1.1.3.38) can also be expressed by host cells to oxidize any residual vanillyl alcohol into vanillin.
  • VAO enzymes are known in the art and include, but are not limited to enzymes from filamentous fungi such as Fusarium moniliformis (GENBANK Accession No. AFJ11909) and Penicillium simplicissium (GENBANK Accession No. P56216; Benen, et al . (1998) J. Biol. Chem. 273:7865-72) and bacteria such as Modestobacter marinus (GENBANK Accession No. YP_006366868) , Rhodococcus jostii (GENBANK Accession No. YP_703243.1) and R. opacus (GENBANK Accession No. EHI39392) .
  • PDC1 and/or glutamate dehydrogenase activity can be reduced.
  • a nucleic acid that inhibits expression of the polypeptide or gene product may be included in a recombinant construct that is transformed into the strain.
  • mutagenesis can be used to generate mutants in genes for which it is desired to inhibit function.
  • levels of products, substrates and intermediates e.g., dehydroshikimic acid, protocatechuic acid, protocatechuic aldehyde, vanillin, and vanillin beta- D-glucoside produced by the recombinant host can be determined by extracting samples from culture media for analysis according to published methods.
  • Recombinant hosts described herein can be used in methods to produce vanillin or vanillin glucoside.
  • the method can include growing the recombinant microorganism in a culture medium under conditions in which vanillin and/or vanillin glucoside biosynthesis genes are expressed.
  • the recombinant microorganism may be grown in a batch, fed batch or continuous process or combinations thereof.
  • the recombinant microorganism is grown in a fermentor at a defined temperature (s) in the presence of a suitable nutrient source, e.g., a carbon source, for a desired period of time to produce a desired amount of vanillin and/or vanillin glucoside.
  • a suitable nutrient source e.g., a carbon source
  • this invention also provides a method for producing vanillin and/or vanillin beta-D-glucoside by providing a recombinant host that produces vanillin and/or vanillin beta-D-glucoside and has reduced production or activity of at least one (or two, three, four, five, six, seven, eight, nine or ten) alcohol dehydrogenase, at least one aldehyde reductase, or at least one alcohol dehydrogenase and at least one aldehyde reductase; cultivating said recombinant host, e.g., in the presence of a suitable carbon source, for a time sufficient for said recombinant host to produce vanillin and/or vanillin glucoside; and isolating vanillin and/or vanillin glucoside from said recombinant host or from the cultivation supernatant.
  • the recombinant host produces a reduced amount of vanillic alcohol in comparison to a host
  • the recombinant host expresses at least one 3DSD and at least one ACAR, which preferably may be one of the 3DSD's and ACAR's described herein.
  • the method may also include determining the level of generated vanillin and iso-vanillin.
  • the recombinant host may also express at least one UGT capable of catalyzing glucosylation of vanillin and isovanillin, in which case the levels of vanillin-glucoside and iso-vanillin-glucoside may be determined instead of the levels of vanillin and iso-vanillin, respectively.
  • this may be determined by generating a recombinant host harboring a heterologous nucleic acid encoding the mutant COMT polypeptide to be tested, and feeding protocatechuic acid to said recombinant host, followed by determining the level of generated iso-vanillic acid and vanillic acid.
  • an in vitro assay or a recombinant host cell can be used to determine whether a mutant COMT polypeptide is capable of catalyzing methylation of an -OH group of protocatechuic aldehyde, wherein said methylation results in generation of at least X times more vanillin compared to iso-vanillin.
  • protecatechuic aldehyde is used as starting material and the level of vanillin and iso-vanillin is determined.
  • an in vitro assay or a recombinant host cell can be used to determine whether a given mutant COMT polypeptide is capable of catalyzing methylation of an -OH group of protocatechuic alcohol, wherein said methylation results in generation of at least X times more vanillyl alcohol compared to iso-vanillyl alcohol.
  • protecatechuic alcohol is used as starting material and the level of vanillyl alcohol and iso-vanillyl alcohol is determined.
  • the level of isovanillin and vanillin may be determined by any suitable method useful for detecting these compounds, wherein said method can distinguish between isovanillin and vanillin. Such methods include for example HPLC.
  • the level of iso-vanillic acid, vanillic acid, iso-vanillyl alcohol and vanillyl alcohol may be determined using any suitable method useful for detecting these compounds, wherein said method can distinguish between isovanillin and vanillin.
  • Such methods include for example HPLC.
  • Carbon sources of use in the method of this invention include any molecule that can be metabolized by the recombinant host cell to facilitate growth and/or production of the vanillin and/or vanillin glucoside.
  • suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, ethanol, glycerol, glucose, cellulose, starch, cellobiose or other glucose containing polymer.
  • sucrose e.g., as found in molasses
  • fructose, xylose, ethanol, glycerol glucose
  • cellulose starch
  • cellobiose or other glucose containing polymer e.g., glucose
  • yeast for example, carbons sources such as sucrose, fructose, xylose, ethanol, glycerol, and glucose are suitable.
  • the carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of
  • vanillin and/or vanillin beta-D-glucoside can then be recovered from the culture using various techniques known in the art, e.g., isolation and purification by extraction, vacuum distillation and multi-stage re-crystallization from aqueous solutions and ultrafiltration (Boddeker, et al . (1997) J. Membrane Sci. 137:155-158; Borges da Silva, et al . (2009) Chem. Eng. Des . 87:1276-1292).
  • Macroporous adsorption resins with crosslinked-polystyrene framework have also been used to recover dissolved vanillin from aqueous solutions (Zhang, et al . (2008) Eur. Food Res. Technol. 226 :377 -383) .
  • Ultrafiltration and membrane contactor (MC) techniques have also been evaluated to recover vanillin (Zabkova, et al . (2007) J " . Membr. Sci. 301:221-237; Scuibba, et al . (2009) Desalination 241:357- 364) .
  • conventional techniques such as percolation or supercritical carbon dioxide extraction and reverse osmosis for concentration could be used.
  • vanillin or vanillin glucoside can be extracted from the plant tissue using various techniques known in the art .
  • vanillin or vanillin beta-D- glucoside can be produced using whole cells that are fed raw materials that contain precursor molecules. The raw materials may be fed during cell growth or after cell growth. The whole cells may be in suspension or immobilized. The whole cells may be in fermentation broth or in a reaction buffer. In some embodiments a permeabilizing agent may be required for efficient transfer of substrate into the cells.
  • the vanillin or vanillin beta- D-glucoside is isolated and purified to homogeneity (e.g., at least 90%, 92%, 94%, 96%, or 98% pure) .
  • the vanillin or vanillin beta-D-glucoside is isolated as an extract from a recombinant host.
  • vanillin or vanillin beta-D-glucoside may be isolated, but not necessarily purified to homogeneity.
  • the amount of vanillin or vanillin beta-D- glucoside produced can be from about 1 mg/1 to about 20,000 mg/L or higher.
  • the recombinant microorganism can be cultured for from 1 day to 7 days, from 1 day to 5 days, from 3 days to 5 days, about 3 days, about 4 days, or about 5 days .
  • Extracts of isolated, and optionally purified, vanillin or vanillin beta-D-glucoside find use in flavoring consumables such as food products, dietary supplements, nutraceuticals , pharmaceutical compositions, dental hygienic compositions, and cosmetic products.
  • the phrase "food product,” as used herein, includes, but is not limited to, fruits, vegetables, juices, meat products such as ham, bacon and sausage; egg products, fruit concentrates, gelatins and gelatin-like products such as jams, jellies, preserves, and the like; milk products such as ice cream, sour cream and sherbet; icings, syrups including molasses; corn, wheat, rye, soybean, oat, rice and barley products, nut meats and nut products, cakes, cookies, confectionaries such as candies, gums, fruit flavored drops, and chocolates, chewing gum, mints, creams, icing, ice cream, pies and breads, beverages such as coffee, tea, carbonated soft drinks, such as COKE and PEPSI, non-carbonated soft drinks, juices and other fruit drinks, sports drinks such as GATORADE, coffee, teas, iced teas, cola, alcoholic beverages, such as beers, wines and liquors, and KO
  • Food products also include condiments such as herbs, spices and seasonings, flavor enhancers.
  • a food product also includes prepared packaged products, such as dietetic sweeteners, liquid sweeteners, granulated flavor mixes which upon reconstitution with water provide non-carbonated drinks, instant pudding mixes, instant coffee and tea, coffee whiteners, malted milk mixes, pet foods, livestock feed, tobacco, and materials for baking applications, such as powdered baking mixes for the preparation of breads, cookies, cakes, pancakes, donuts and the like.
  • Food products also include diet or low-calorie food and beverages containing little or no sucrose. Other examples of food products envisioned in accordance with the present invention are described below and throughout the specification .
  • the food products are fruits, vegetables, juices, meat products such as ham, bacon and sausage; egg products, fruit concentrates, gelatins and gelatin-like products such as jams, jellies, preserves, and the like; milk products such as ice cream, sour cream and sherbet; icings, syrups including molasses; corn, wheat, rye, soybean, oat, rice and barley products, nut meats and nut products, cakes, cookies, confectionaries such as candies, gums, fruit flavored drops, and chocolates, creams, icing, ice cream, pies and breads.
  • the consumable is a pharmaceutical composition.
  • Preferred compositions are pharmaceutical compositions containing vanillin and/or vanillin beta-D-glucoside and one or more pharmaceutically acceptable excipients. These pharmaceutical compositions can be used to formulate pharmaceutical drugs containing one or more active agents that exert a biological effect. As such, the pharmaceutical composition preferably further include one or more active agents that exert a biological effect. Such active agents include pharmaceutical and biological agents that have an activity. Such active agents are well known in the art. See, e.g., The Physician's Desk Reference. Such compositions can be prepared according to procedures known in the art, for example, as described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, USA.
  • such an active agent includes bronchodilators , anorexiants, antihistamines, nutritional supplements, laxatives, analgesics, anesthetics, antacids, H2 -receptor antagonists, anticholinergics, antidiarrheals , demulcents, antitussives, antinauseants , antimicrobials, antibacterials , antifungals, antivirals, expectorants, anti- inflammatory agents, antipyretics, and mixtures thereof.
  • the active agent is an antipyretics or analgesics, e.g., ibuprofen, acetaminophen, or aspirin; laxatives, e.g., phenolphthalein dioctyl sodium sulfosuccinate ; appetite depressants, e.g., amphetamines, phenylpropanolamine, phenylpropanolamine hydrochloride, or caffeine; antacidics, e.g., calcium carbonate; antiasthmatics, e.g., theophylline; antidiuretics, e.g., diphenoxylate hydrochloride; agents active against flatulence, e.g., simethecon; migraine agents, e.g., ergotaminetartrate ; psychopharmacological agents, e.g., haloperidol ; spasmolytics or sedatives, e.g., phenobar
  • Active substances which have a particularly unpleasant taste include antibacterial agents such as ciprofloxacin, ofloxacin, and pefloxacin; antiepileptics such as zonisamide; macrolide antibiotics such as erythromycin; beta-lactam antibiotics such as penicillins and cephalosporins; psychotropic active substances such as chlorpromazine ; active substances such as sulpyrine; and agents active against ulcers, such as cimetidine.
  • antibacterial agents such as ciprofloxacin, ofloxacin, and pefloxacin
  • antiepileptics such as zonisamide
  • macrolide antibiotics such as erythromycin
  • beta-lactam antibiotics such as penicillins and cephalosporins
  • psychotropic active substances such as chlorpromazine
  • active substances such as sulpyrine
  • agents active against ulcers such as cimetidine.
  • compositions of this invention are administered to a subject in any form suitable to achieve their intended purpose.
  • the composition is one which can be administered buccally or orally.
  • the pharmaceutical composition can be an oral or nasal spray.
  • the subject is any animal, such as a human, although the invention is not intended to be so limited.
  • suitable animals include canines, felines, dogs, cats, livestock, horses, cattle, sheep, and the like.
  • a veterinary composition, as used herein, refers to a pharmaceutical composition that suitable for non-human animals. Such veterinary compositions are known in the art.
  • the pharmaceutical composition is a liquid dosage form for oral administration, including pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms can contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and
  • Suspensions in addition to the active compounds, can contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.
  • the pharmaceutical composition of the present invention can be in the form of a chewable tablet.
  • Chewable tablets are known in the art. See, e.g., U.S. Patent Nos . 4,684,534 and 6,060,078, each of which is incorporated by reference in its entirety. Any kind of medicament can be contained in the chewable tablet, preferably a medicament of bitter taste, natural plant extracts or other organic compounds .
  • vitamins such as vitamin A, vitamin B, vitamin Bl, vitamin B2 , vitamin B6, vitamin C, vitamin E and vitamin K; natural plant extracts such as Sohgunjung-tang extracts, Sipchundaebo-tang extracts and Eleutherococcus senticosus extracts; organic compounds such as dimenhydrinate , meclazine, acetaminophen, aspirin, phenylpropanolamine, and cetylpyridinium chloride; or gastrointestinal agents such as dried aluminum hydroxide gel, domperidone, soluble azulene, L-glutamine and hydrotalcite can be contained in the core.
  • the pharmaceutical composition of the present invention can be an orally disintegrating composition.
  • Orally disintegrating tablets are known in the art. See, e.g., U.S. Patent Nos . 6,368,625 and 6,316,029, each of which is hereby incorporated by reference in its entirety.
  • the pharmaceutical composition of the present invention can be a solid dosage form, including vanillin or vanillin beta-D-glucoside and a water and/or saliva activated effervescent granule, such as one having a controllable rate of effervescence.
  • the effervescent composition can further comprise a pharmaceutically active compound.
  • Effervescent pharmaceutical compositions are known in the art. See, e.g., U.S. Patent No. 6,649,186, which is incorporated by reference in its entirety.
  • the effervescent composition can be used in pharmaceutical, veterinary, horticultural, household, food, culinary, pesticidal, agricultural, cosmetic, herbicidal, industrial, cleansing, confectionery and flavoring applications.
  • Formulations incorporating the effervescent composition containing vanillin or vanillin beta-D-glucoside can further include one or more additional adjuvants and/or active ingredients which can be chosen from those known in the art, including flavors, diluents, colors, binders, filler, surfactant, disintegrant , stabilizer, compaction vehicles, and non-effervescent disxntegrants.
  • the pharmaceutical composition can be a film-shaped or wafer- shaped pharmaceutical composition.
  • a film- shaped or wafer-shaped pharmaceutical composition can be configured, for example, as quickly disintegrating administration forms, e.g., administration forms disintegrating within a period of 1 second up to 3 minutes, or as slowly disintegrating administration forms, e.g., administration forms disintegrating within a period of 3 to 15 minutes.
  • the indicated disintegration times can be set to the above-mentioned ranges by using, for example, matrix- forming polymers which have different disintegrating, or solubility, characteristics.
  • the disintegration time can be adjusted.
  • disintegrants are known which "draw" water into the matrix and cause the matrix to burst open from within.
  • certain embodiments of the invention include such disintegrants for the purpose of adjusting the disintegration time.
  • Suitable are polymers for use in the film-shaped or wafer-shaped pharmaceutical composition include cellulose derivatives, polyvinyl alcohol (e.g. MOWIOL) , polyacrylates , polyvinyl pyrrolidone, cellulose ethers, such as ethyl cellulose, as well as polyvinyl alcohol, polyurethane, polymethacrylates , polymethyl methacrylates and derivatives and copolymer! zates of the aforementioned polymers .
  • the total thickness of the film- shaped or wafer- shaped pharmaceutical composition according to the invention is preferably 5 ⁇ up to 10 mm, preferably 30 ⁇ to 2 mm, and with particular preference 0.1 mm to 1 mm.
  • the pharmaceutical preparations can be round, oval, elliptic, triangular, quadrangular or polygonal shape, but they can also have any rounded shape.
  • the pharmaceutical composition of the present invention can be in the form of an aerosol .
  • the aerosol composition can further include a pharmaceutically active agent. Aerosol compositions are known in the art. See, e.g., U.S. Patent No. 5,011,678, which is hereby incorporated by reference in its entirety.
  • an aerosol composition according to the present invention can include a medically effective amount of a pharmaceutically active substance, vanillin or vanillin beta-D-glucoside and a biocompatible propellant, such as a (hydro/fluoro) carbon propellant.
  • the pharmaceutical composition is a nutritional composition.
  • nutritional compositions having an undesirable taste include, but are not necessarily limited to, enteral nutrition products for treatment of nutritional deficit, trauma, surgery, Crohn's disease, renal disease, hypertension, obesity and the like, to promote athletic performance, muscle enhancement or general well being or inborn errors of metabolism such as phenylketonuria.
  • enteral nutrition products for treatment of nutritional deficit, trauma, surgery, Crohn's disease, renal disease, hypertension, obesity and the like, to promote athletic performance, muscle enhancement or general well being or inborn errors of metabolism such as phenylketonuria.
  • such nutritional formulations can contain one or more amino acids which have a bitter or metallic taste or aftertaste.
  • amino acids include, but are not limited to, an essential amino acids such as an L isomer of leucine, isoleucine, histidine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine or valine.
  • an essential amino acids such as an L isomer of leucine, isoleucine, histidine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine or valine.
  • the consumable of the present invention is a dental hygienic composition containing vanillin and/or vanillin beta-D-glucoside.
  • Dental hygienic compositions are known in the art and include, but are not necessarily limited to, toothpaste, mouthwash, plaque rinse, dental floss, dental pain relievers (such as ANBESOL) , and the like.
  • the consumable of the present invention is a cosmetic product containing vanillin and/or vanillin beta-D-glucoside .
  • the cosmetic product can be a face cream, lipstick, lip gloss, and the like.
  • suitable compositions of the invention include lip balm, such as CHAPSTICK or BURT'S BEESWAX Lip Balm, further containing vanillin and/or vanillin beta-D-glucoside.
  • Example 1 Yeast Strains with Reduced Alcohol Dehydrogenase/Aldehyde Reductase Activity
  • Yeast strains lacking alcohol dehydrogenase and/or aldehyde reductase were produced in a parent strain of yeast that was capable of producing vanillin/vanillin beta- D-glucoside.
  • the parent strain, EFSC2932 was created by integrating exogenous OMT, UGT and 3DSD genes into the genome of strain EFSC2055 (Genotype: Mata his3Dl leu2D0 metl5D0 ura3D0 adh6 : : LEU2 bgll : : KanMX4
  • PTPI1 :3DSD[AurC] : : (HsOMT: :MET15 [NatMX] ) : : ACAR [HphMX] : :UGT7 2E2[HIS3] ECM3 : : (CorPPTase-ScHAP4) .
  • Genes encoding OMT, UGT and 3DSD were amplified by polymerase chain reaction (PCR) with gene-specific primers using X7 DNA polymerase.
  • Vectors for chromosomal integration of gene expression were constructed using the uracil -specific excision reagent
  • Candidate genes encoding alcohol dehydrogenases or aldehyde reductases were selected and knocked out in various combinations. Cultures of each knockout were analyzed using HPLC-UV to quantify vanillin glucoside/isovanillin glucoside and related products. HPLC analysis was carried out with an AGILENT 1100 series system with binary pump and a Phenomenex Synergi Polar-RP 2.5u 100A 100x2.00mm column, which separates precursors and Isovanillin and vanillin. A flat gradient was run with water/acetonitrile + 0.1% trifluoroacetic acid. A 8.9 minute program + 1.1 minute postrun was carried out as presented in Table 2.
  • Vanillin glucoside and isovanillin glucoside were quantified by integrating the area of the HPLC peaks and comparing the same with a standard curve.
  • the combinations of genes disrupted and the resulting effect on vanillyl alcohol production are provided in Table 3.

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Abstract

L'invention concerne des microorganismes, plantes et cellules végétales recombinés, qui ont été modifiés de manière à présenter des taux réduits ou une activité réduite d'un ou plusieurs alcool déshydrogénases ou aldéhyde réductases, de manière à accroître la production de vanilline ou de bêta-D-glucoside vanillique.
PCT/US2014/046315 2013-07-16 2014-07-11 Compositions et procédés de biosynthèse de vanilline ou d'un bêta-d-glucoside de vanilline WO2015009558A1 (fr)

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EP14826331.2A EP3022291B1 (fr) 2013-07-16 2014-07-11 Compositions et procédés de biosynthèse de vanilline ou d'un bêta-d-glucoside de vanilline
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JP2019531759A (ja) * 2016-10-26 2019-11-07 味の素株式会社 目的物質の製造方法
EP3487318A4 (fr) * 2016-07-19 2020-04-08 Pepsico, Inc. Analogues de l'acide glucosyringique en tant que modificateurs du profil édulcorant
WO2021022216A1 (fr) * 2019-08-01 2021-02-04 Amyris, Inc. Cellules hôtes modifiées pour la production à haut rendement de vanilline
CN113201465A (zh) * 2021-05-07 2021-08-03 天津大学 酿酒酵母工程菌及其在制备香草醛中的应用
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WO2015121379A3 (fr) * 2014-02-12 2015-10-08 Evolva Sa Méthodes d'amélioration de la production de vanilline
WO2017073701A3 (fr) * 2015-10-27 2017-08-10 Ajinomoto Co., Inc. Procédé de production d'aldéhyde
CN108350412A (zh) * 2015-10-27 2018-07-31 味之素株式会社 用于生产醛的方法
JP2018531617A (ja) * 2015-10-27 2018-11-01 味の素株式会社 アルデヒドの製造方法
CN108350412B (zh) * 2015-10-27 2022-02-11 味之素株式会社 用于生产醛的方法
US10612006B2 (en) 2015-10-27 2020-04-07 Ajinomoto Co., Inc. Method for producing aldehyde
US10966447B2 (en) 2016-07-19 2021-04-06 Pepsico, Inc. Glucosyringic acid analogs as sweetness profile modifiers
AU2017299518B2 (en) * 2016-07-19 2022-03-03 Pepsico, Inc. Glucosyringic acid analogs as sweetness profile modifiers
EP3487318A4 (fr) * 2016-07-19 2020-04-08 Pepsico, Inc. Analogues de l'acide glucosyringique en tant que modificateurs du profil édulcorant
JP2019531759A (ja) * 2016-10-26 2019-11-07 味の素株式会社 目的物質の製造方法
CN109890960A (zh) * 2016-10-27 2019-06-14 味之素株式会社 用于生产醛的方法
US10883125B2 (en) 2016-10-27 2021-01-05 Ajinomoto Co., Inc. Method for producing aldehyde
JP2019536450A (ja) * 2016-10-27 2019-12-19 味の素株式会社 アルデヒドの製造方法
WO2018079705A1 (fr) * 2016-10-27 2018-05-03 Ajinomoto Co., Inc. Procédé de production d'aldéhyde
JP2022140514A (ja) * 2016-10-27 2022-09-26 味の素株式会社 アルデヒドの製造方法
CN109890960B (zh) * 2016-10-27 2023-02-17 味之素株式会社 用于生产醛的方法
JP7380768B2 (ja) 2016-10-27 2023-11-15 味の素株式会社 アルデヒドの製造方法
WO2021022216A1 (fr) * 2019-08-01 2021-02-04 Amyris, Inc. Cellules hôtes modifiées pour la production à haut rendement de vanilline
CN113201465A (zh) * 2021-05-07 2021-08-03 天津大学 酿酒酵母工程菌及其在制备香草醛中的应用
CN113201465B (zh) * 2021-05-07 2023-01-20 天津大学 酿酒酵母工程菌及其在制备香草醛中的应用
WO2023159017A1 (fr) 2022-02-15 2023-08-24 Evolva Sa Procédé de récupération et de purification de vanilline

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